Orofacial clefting (primarily cleft lip and/or cleft palate) is a relatively common structural birth defect with environmental and genetic contributions to etiology. Genome wide association studies (GWAS) and linkage studies have identified many gene variants that are associated with elevated risk for isolated oral facial clefting (OFC). However, our understanding of the pathogenic mechanisms underlying this disease remains poor because, one, we have yet to distinguish DNA variants that directly influence risk for OFC (i.e., causal variants) from those that are merely in linkage disequilibrium with them, and two, the functions of the regulatory molecules encoded y OFC-associated genes in craniofacial development are largely unknown. At each locus, there are multiple identified multiple SNPs that are statistically associated with OFC ? and all of these reside in non-coding DNA. In Aim 1, we will prioritize the SNPs for functional tests by performing fine mapping of GWAS data, and identifying de novo mutations in new whole genome sequence data from 800 case-parent trios. In Aim 2 we propose to identify the OFC-associated SNPs that are functional (causal). We hypothesize that pathogenic SNPs reside in enhancers that drive expression in oral tissues, and that risk alleles of such SNPs quantitatively affect activity of the enhancers. To test this hypothesis, we will amplify genomic DNA containing risk-associated SNPs and test them for allele-dependent enhancer activity in vitro (cell-based reporter assays). We will also test the tissue specificity of the enhancers (zebrafish and mouse-based reporter assays). In Aim 3, we will determine the effect that altering the allele of pathogenic SNPs has on expression of the relevant OFC-risk gene (genome engineering with CRISPR/Cas9 in vitro). Finally, we apply chromatin immuno precipitation and chromatin configuration capture in the oral epithelium cell line to deduce the mechanism by which functional SNPs change expression of the OFC-risk genes. The expected outcome of the proposed experiments is identification of the mechanisms by which genetic risk variants cause a common birth defect.